Genetic inheritance material of a living organism (DNA) is stored in chromosomes. Understanding of cellular activities in normal development and disease requires more than the DNA sequence and individual- specific variations of the genome. Critical regulatory roles are also played by packaging of the genome into a conserved nucleoprotein complex termed chromatin. Chromatin is assembled and maintained by a concerted action of two families of proteins, namely histone chaperones and ATP-driven motor chromatin remodeling factors. The structure of chromatin and the regulatory machinery of its metabolism are uniquely important and thus strongly conserved in evolution. The ultimate objective of our work is to understand the relationship between the establishment of chromatin structure and regulation of the function of eukaryotic chromosomes. We will focus on a systematic study of proteins that mediate chromatin assembly. We will analyze elementary molecular events and examine interactions at the interface of histone chaperones and motor factors that take place during chromatin assembly in vitro (in a test tube). In addition, we will investigate specific functional roles for chromatin assembly factors in a living model organism, fruit fly (Drosophila melanogaster). We will perform experiments to understand how the assembly factors alter chromatin structure in vivo and regulate various DNA-directed enzymatic processes, such as transcription, DNA replication and repair. The chromatin of mature sperm differs in composition from normal cell chromatin, in particular by an extraordinary high degree of DNA condensation. Sperm chromatin is enzymatically static and is formed by compaction of DNA with small basic, cysteine-rich proteins termed protamines. At fertilization, the egg faces the challenge of remodeling the condensed sperm chromatin into an accessible, transcription- and replication- competent form. We recently discovered cellular machinery that mediates sperm chromatin remodeling in vitro and in vivo. Thus, we will also study the factors that mediate sperm chromatin remodeling. The successful completion of this project will lead to a comprehensive biochemical and biological characterization of factors that mediate the assembly of various forms of chromatin. More globally, our work will provide insights into the role of chromosome assembly and maintenance in regulation of the cell function and will be applicable to understanding, diagnosis and treatment of human diseases that involve defects in processes of DNA metabolism.